WO1992002619A1 - Plasmide destine a la production d'une proteine inhibant la phospholipase a2 derivee d'une region enflammee, cellule microbienne, et production et utilisation de ladite proteine - Google Patents

Plasmide destine a la production d'une proteine inhibant la phospholipase a2 derivee d'une region enflammee, cellule microbienne, et production et utilisation de ladite proteine Download PDF

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Publication number
WO1992002619A1
WO1992002619A1 PCT/JP1991/001040 JP9101040W WO9202619A1 WO 1992002619 A1 WO1992002619 A1 WO 1992002619A1 JP 9101040 W JP9101040 W JP 9101040W WO 9202619 A1 WO9202619 A1 WO 9202619A1
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Prior art keywords
protein
plasmid
dna
fragment
inhibitory
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PCT/JP1991/001040
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English (en)
French (fr)
Japanese (ja)
Inventor
Yorimasa Suwa
Atsushi Imaizumi
Masahiro Okada
Yoji Suzuki
Ichiro Kudo
Keizo Inoue
Chieko Azuma
Makoto Murakami
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Teijin Limited
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Priority to KR1019920700763A priority Critical patent/KR920702418A/ko
Publication of WO1992002619A1 publication Critical patent/WO1992002619A1/ja

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is inflammatory sites derived phospholipase A z inhibitory protein recombinant plasmid having a DNA encoding a fragment, the plasmid recombinant microorganism transformed with de, inflammation locally derived phospho "lipase A 2 using the microorganism
  • the present invention also relates to a method for producing a protein by the expression of an inhibitory protein gene, and the present invention also relates to an allergic reaction inhibitor comprising the protein produced by the method as an active ingredient.
  • Complement C 3 (hereinafter referred to as C 3) is a protein known to play a central function in the complement activation pathway. This C3 is gradually degraded by proteolytic enzymes in the blood. That is, it is first cleaved by C3 complementase to produce C3a and C3b. C 3 b binds to the foreign body surface via a thiol ester site, which subsequently activates the complement pathway leading to the formation of a membrane attack complex. In addition, C 3 a acts as anaphyla toxin. C3b is then further cleaved by proteolytic enzymes to ultimately yield C3dg or C3d.
  • Phospho lipase A 2 is hydrolyzed worth the ⁇ Esuteru binding of Li phospholipids It is an enzyme that breaks down and produces fatty acids and lysolin lipids. In particular, arachidonic acid, a precursor of prostaglandin, leukotriene, thromboxane, etc., is released from membrane phospholipids and plays an important role in the production of these inflammatory mediators. it is conceivable that. Recently, the Hosuhoribaze eight 2 from local human inflammatory diseases and inflammatory animal model purified, its properties were clearly summer. This enzyme is considered to have an action to promote the inflammatory response, and therefore, a drug that inhibits the activity of this enzyme is expected to exhibit an anti-inflammatory effect.
  • the present inventors have focused quickly to the enzyme, which specifically perform exploratory research of proteins that inhibit, C 3 dg of human and rats to specifically inhibit inflammation localized derived Hosuhoribaze A 2 activity from having sex, C 3 dg inhibits Hosuhori lipase a 2 in inflammatory sites, as a result may act anti-inflammatory manner has already filed found that there (Japanese Patent application No. 1 one 200 246 No. And Japanese Patent Application No. 2 — 89085, these applications correspond to WO 91/01 999 published internationally after the application for Japanese Patent Application No. 205164, which is the basis of the priority claim of the present application. ).
  • C3dg in order to evaluate the anti-inflammatory effect of C3dg and to develop it as an anti-inflammatory drug in the future, it is necessary to obtain a large amount of purified protein.
  • Conventionally known methods for obtaining C3 dg include treating serum with 37, or treating purified C3 with a proteolytic enzyme such as Factor I, followed by various methods.
  • the present inventors have conducted intensive studies to solve such problems, and as a result, by using a gene recombination technique, the C3 dg portion of rats and humans was inserted, and phospholipase equivalent to C3 dg was obtained. and finding a method of producing large amounts of protein with a 2 inhibitory activity, thus completing the present onset bright.
  • the protein of the present invention is interested as a prophylactic or therapeutic drug for more specific diseases Disease and specifically inhibit child inflammation localized derived phospholipase A z.
  • the protein of the present invention is interested as a prophylactic or therapeutic drug for more specific diseases Disease and specifically inhibit child inflammation localized derived phospholipase A z.
  • the present invention provides a plasmid comprising a DNA sequence encoding a local inflammatory phospholipase 2 inhibitory protein and a DNA sequence having one function of a promoter that regulates the expression of the protein.
  • transformed recombinant microorganism cells, further the recombinant microorganism were cultured in a nutrient medium until a inflammation localized derived Hosuhoripa over peptidase a 2 inhibitory proteins, the inflammatory sites derived phospholipase a 2 inhibitory protein from the culture Yobutsu inflammatory sites derived phospholipase a 2 inhibition process for producing a protein, characterized by preparative adopted, as well as to provide inflammatory topical derived phospholipase a 2 inhibitory proteins produced by the production method.
  • inhibitory protein, or mammalian organs also properly Izu from the body fluid of isolated and purified or inflammatory sites from Complement C 3 mammalian been decomposed prepared from phospholipase eight 2 11 harm agent
  • the present invention provides an allergic reaction inhibitor.
  • FIG. 1 is a schematic diagram showing the construction of a rat C 3 or fusion protein expression plasmid.
  • Figure 2 (a) and (b) are schematic diagrams of the construction of the Rat Met-C3dg expression plasmid.
  • Fig. 3 (a) and (b) are schematic diagrams of the construction of a human Met-C3dg expression brasmid.
  • Fig. 4 (a) and (b) show the protein elution peak of the gel filtration HPLC fraction and the SDS of the elution fraction in Example 5. Indicates PAG.
  • Figure 5 shows a rat inflammatory sites-derived Hosuhoribaze A 2 inhibitory activity of the fusion protein in Example 7.
  • FIG. 6 shows the histamine release rate (%) of the fusion protein in Example 8.
  • the symbol indicates the control, and the symbol indicates the fusion protein.
  • a DNA sequence that encodes a protein is an example of the entire amino acid K sequence of rat or human C 3 dg or the DNA sequence encoding them.
  • PC TZ JP90 / 0996 As described in WO 91/0199 9 published internationally after filing as the basis of the priority of this patent application (Japanese Patent Application No. 205205, filed on August 3, 1990) Have been. The contents are incorporated herein by reference.
  • Such amino acid sequences are illustrated by SEQ ID NOs: 1 and 3.
  • An example of a DNA sequence encoding such an amino acid sequence is specifically shown in SEQ ID NOs: 2 and 4. And those derived from rat and human, each consisting of 10.32 and 10.47 bases, respectively.
  • SEQ ID NOs: 2 and 4 and those derived from rat and human, each consisting of 10.32 and 10.47 bases, respectively.
  • the Hosuhoriba one peptidase A 2 inhibitory proteins produced by the expression is not lost substantially its activity, its upstream contact and /
  • Those with an additional DNA sequence downstream are also included in the DNA sequence encoding the inhibitory protein.
  • Additional DNA sequences are generally those that may accompany the cloning of the DNA sequence of interest.
  • a rat sequence prepared in the process of constructing a plasmid as exemplified in FIGS. Enclosed by a DNA sequence that encodes a phosphorylase 2 inhibitory protein.
  • a DNA sequence that encodes a phosphorylase 2 inhibitory protein include a 2.1 kbp DNA fragment derived from a rat liver cDNA library gt11 library, wherein the DNA fragment is a cDNA ⁇ gt11 library.
  • a double-stranded DNA prepared by ligating double-stranded DNA prepared by connecting about 60 bp, and further connecting a termination codon and about 20 bp of the 3 'end of the rat C 3 dg gene to the downstream region DNA sequence reconstructed by ligating DNA And 1.2 kb Eco R obtained by digesting human cDNA clone PHLC 3.11 with Eco RI and Kpn I.
  • Nc of double-stranded DNA prepared by connecting the start codon and about 70 bp of the 5 'end of the C3dg gene to the upstream region of the I-KpnI fragment.
  • PLA 2 ⁇ DNA is DNA encoding sequence of PLA 2 inhibitory protein
  • end is a stop codon
  • a signal 'DNA will code the signal peptide de sul D a NA sequence
  • a 'D NA Koh additional poly peptide to express large amounts of PLA 2 inhibitory protein in a host cell
  • DNA is the DNA sequence encoding the cleavage sequence peptide.
  • the DNA sequence having a promoter function for regulating the expression of the protein includes lactose operon.
  • lac lac
  • tributophan (trp) promoter trp-1 ac hybrid promoter (eg, tac, tacII, trc), sphage P L promoter and P R promoter, T 7 promoter, Te door Rasai click Li down resistance gene (tet) promoter, trp - tet and 1 ac - tet Nono Ivry-head promoter, ⁇ - Rakutamazepuro motor, E. coli outer membrane protein gene (1PP) promoters, synthetic promoters (eg, E. coli trp promoter, 82-base DNA containing an operator) and the like.
  • the 1 ac promoter can be particularly preferred.
  • Vectors that can be used for preparing the plasmid of the present invention include those that are known per se or commercially available vectors as they are, or those that are derived according to the intended use. .
  • an expression vector in which elements such as the above-mentioned promoter sequence, Shine-Dalgarno (SD) sequence, and a terminator are incorporated in advance can be conveniently used.
  • Other elements that may be incorporated include a DNA sequence that encodes the above signal peptide, for example, When Escherichia coli is used as a host, alkaline phosphatase
  • DNA sequences encoding signal peptides such as OmpF, protein A, ⁇ -lactamase (polysilase), riboprotein and endotoxin.
  • Examples of expression plasmids in Escherichia coli include P01 series plasmids such as PBR series and pUC series, P15A series plasmids such as pACYC series, mini F vector, etc.
  • Examples include F factor-based plasmids, R-factor-based plasmids such as PSC101, and phage-derived plasmids such as pUC118 and 119.
  • PTV118N, pTV119N, etc. which contain the lac promoter, SD sequence, translation initiation codon, cloung site, and terminator in this order.
  • brass Mi de of the present invention are those comprising a click Roningusai bets on PLA 2 inhibitory protein before Symbol vector DNA encoding sequence in I Complex is ⁇ up are, like correct ones Examples are PPTC 3 Z2.1, 3 ⁇ 413 ⁇ 43 / 1.035 and PTHC3 / 1.500. These brassmids are recombinant microbial cells obtained by transforming Eschericia coli JM109 using the same, and are sent to the Patent Microorganisms Depositary of the Institute of Microbial Engineering, the Ministry of International Trade and Industry of Japan.
  • FERMBP—3 027 (transferred from FERMP—1 1 3 4 1 deposited on April 26, 1990 to international deposit), FERMBP—3 4 8 5 (1991 Deposited on July 17) and FERMBP— 3 4 8 6 (1 9 9 1 E. coli, deposited on July 17, 2007), which can be obtained from PTC3Z2.1, J9TRC3 / 1.035 and J9THC3 / 1.050 strains, respectively, by a conventional method.
  • Burasumi de of the present invention including these plasmids, the insertion of the DNA sequence encoding the PLA 2 inhibitory protein to the claw Jung site of the above vector, for example the 1, 2 (a) (b and 3 ( a) (b) It can be prepared according to any of the plasmid construction procedures shown in the figures, and each of these steps can be carried out by a method known per se.
  • the recombinant microbial cell of the present invention can be obtained by transforming the microbial cell with the brasside obtained as described above. It is good preferable to use a C a C 1 2 methods upon this transformation.
  • examples of the host cell for recombination include microbial cells belonging to the genus Eschericia, the genus Bacillus, the genus Saccharomyces, and the like. Among these, microorganism cells of the genus Eschericia, and particularly, Eschericia coli JM109 are preferable.
  • recombinant microbial cells of the present invention include Escherichia coli PTC32.1 (FERMBP-307) and J9TRC3 transformed with the above-described preferred plasmid of the present invention. /1.035 (FERMBP—3485) and J 9 THC 3 /1.050 (FERMBP—3486).
  • the above recombinant microbial cells can be used for production of a protein having a phospholipase 2 inhibitory activity derived from local inflammation.
  • the present invention also the recombinant microorganism cells are cultured in nutrient culture place to produce inflammation locally derived Hosuhoribaze A 2 inhibitory protein (PLA 2 inhibition protein), collecting PLA 2 inhibition ⁇ white from the culture
  • PLA 2 inhibition protein Hosuhoribaze A 2 inhibitory protein
  • the cultivation is carried out under the conditions usually used for culturing E. coli, and the nutrient medium can also be carried out using ordinary carbon sources, nitrogen sources, other inorganic salts and micronutrients. This to PLA 2 inhibition ⁇ white, either large accumulation as inclusion bodies in bacterial cells, and is accumulated in the use the extracellular DNA sequences encoding the signal peptide de as described above.
  • P LA Z inhibitory protein from such cultures, in the former case, appropriate lysis treatment in the microbial cells, for example after lysed by subjecting the cells Kabe ⁇ hydrolase treatment or sonication, also latter In this case, after the cells are removed, a protein separation and purification method known per se may be performed. More specifically, in the former, the collected cells are collected by, for example, sonication or the like, and the inclusion body is recovered, the inclusion body is solubilized with urea, etc., and urea is removed using a gel filtration ram. Pyridylethylation of proteins protects SH groups.
  • the SH-protected protein is fractionated by gel filtration HPLC to obtain a fraction containing a recombinant protein derived from the C3 ⁇ gene as a main component.
  • the protein is further isolated and purified by reverse phase HPLC or the like.
  • the inhibitory activity or inhibitory protein can be measured or tracked as follows, for example.
  • Casein derived rats peritoneal exudate than isolated as an enzyme - using phospholipase A 2 was purified as the substrate "C - extracted from E.Coli cultured with acetic acid, 14 C one-phospha Chi Gilles ethanolamine Mi purified (About 2,000 dpm / nmol, 0.1 mM).
  • the protein was transferred from the gel to a two-nitrocellulose filter using a Millipore Electro-Plotting Apparatus, and anti-human C 3d hidge serum (Dako), horseradish Using a peroxidase-conjugated anti-hidden IgG antibody (Cappel) and 4-substituted 11-naphthol (Bi0-Rad) as the substrate, a C3d band was detected by enzyme-antibody staining. Can be detected.
  • inflammation locally derived phospho lipase A 2 inhibitory proteins produced by, for example, a protein consisting of I Complex the amino acid sequence shown SEQ ID NO: 1 or 3 as a main sequence, inflammation locally derived phospholipase as described below which has specifically strong inhibitory activity against eight 2, it was confirmed that even allergic reaction inhibition.
  • inflammatory topical derived phospholipase A z specifically inhibitory protein that is is known to be, for example, JP-63- described in JP 246 397 rats intraperitoneally derived proteins
  • Such effects are based on the finding that these inhibitory proteins, surprisingly, inhibit mast cell-derived reliance on granules. Therefore, another aspect of the present invention relates to A Rerugi first reaction inhibitor comprising at I Complex inflammation localized derived phospholipase A 2 inhibitory protein as an active ingredient.
  • the inhibitory protein include those obtained by the genetic method of the present invention, Also included are those that are isolated and purified, and those that are prepared from trap C3.
  • the allergic reaction in the present invention refers to a reaction that occurs when an antigen-sensitized living body again receives an allogeneic antigen. Above all, an immediate type such as an anaphylaxis or an aruss reaction induced by mast cells. An allergic reaction. Specific diseases of the mammals of interest, particularly humans, include asthma and measles.
  • the PLA 2 inhibitory protein as an active ingredient, soft capsules using suitable excipients and the like in a known manner, hard capsules, tablets, oral agents such as white-up, injection Or can be used as an external preparation.
  • the dose of the active ingredient is usually about 1 to 50 Omg per day, and the number of doses is usually 1 to 3 times / day.It is necessary to adjust the formulation so as to satisfy such conditions. preferable.
  • the active ingredient does not show acute toxicity to mammals within this administration range.
  • the inhibitor of the present invention can be used in combination with an existing drug.
  • the abdominal cavity of a male Wistar rat (weighing more than 500 g) was injected with 50 ml of Hank's solution containing 10 ml of hebarin,
  • the obtained peritoneal mast cells were resuspended in Hank's solution containing 0.1% gelatin (5 ⁇ 10 b i il), and 1 / ml of anti-DNPI gE antibody (Miles) was added. And passively sensitize the cells.
  • the cells are washed and resuspended in a Hanks solution containing 0.1% gelatin to a cell concentration of 12 ⁇ 10 6 ml.
  • an appropriate amount of the antigen, DNP-I Ascharis (prepared by the method described in the method of Eisen et al., J. Am. Chem.
  • HMT histamine N-methyltransferase
  • the kidney of the SD rat (200 g) is homogenized in 9 times the wet weight of 0.25 M sucrose solution, and centrifuged at 40,000 xg for 1 hour. Add ammonium sulfate to the supernatant, and collect the 45% -70% ammonium sulfate precipitated fraction. Dissolved in 0.1 M phosphate buffer (PH7.4) Then, the plate is folded with respect to 0.01 M phosphate buffer (PH 4). Use this sample as HMT.
  • Hisuta take Mi down the Complex free samples of (1 9 one 5 0) in the test tube, where a suitable amount of HMT and 1 of 3 H (Application Benefits Chi ⁇ beam) labeled S- adeno Shirume Chionin (New England Nuclear) And adjust the total volume to 200 W with 0.1 M phosphate buffer (PH7.9). 3 7 'after Lee Nkyube preparative 9 0 min C, 1 0 0 W 2.5 added to stop the reaction NN a 0 H, further addition of chloroform 2 ml, resulting 3 H- main Chiruhisuta Mi emissions Is extracted into the chloroform layer.
  • Example 1 (Sansharei) rat inflammation cloning topical derived phospholipase A 2 inhibitory proteins Yadenko
  • Rat liver cDNA library gt11 Library (manufactured by Clontech) was used as a screening material, and screens were prepared according to the description of the Clontech experimental protocol. In other words, about 50,000 l of transformants obtained by culturing E.co1iY1909 strain infected with Sgt11 phage for 37 hours for 5 hours were used. Replicate to a lonfil membrane and apply to 0.5 M NaOH-1.5 NaCl to denature the DNA. Tris-HCl buffer containing 1.5 MNaC1-1 MMEDTA (PH 7. 2) Neutralized. After that, air-dried off I Luther, was fixed by irradiation with ultraviolet rays for 3 minutes at the door lance illuminator DNA into full I Luther ⁇
  • a mouse C3c DNA fragment obtained in the above (A) labeled with 32 P was used as the screening probe.
  • the group of transformants on the test filter was screened with this probe by 65 hybridizations and judged by exposure to autoradiogram. Seven of them were positive clones. These positive clones were named rC3 / 11 to 17 respectively.
  • the 0.1 kbp fragment and the 0.6 kbp fragment obtained by BamHI digestion were further introduced into the double digest of EcoRI and BamHI of the sequencing vector PUC118.
  • the nucleotide sequence was determined from the end.
  • the E. coli expression vector PTV119N (Takara Shuzo) was digested with BamHI and EcoRI.
  • Rat C3c DNA clone PTC3Z11 was digested with BamHI and EcoRI, followed by separation of the purified fragment by agarose gel electrophoresis, and 0.1 kb BamHI-EcoRI. The fragment and the EcoRI single EcoRI fragment of .0 kb were recovered.
  • a ligation reaction was performed on the digested vector and the 0.1 kb fragment, and E. coli JM109 was transformed. After that, the transformant was cultured on ambicillate to obtain a transformant.
  • the plasmid, DNA, was extracted and purified from the Transformant, and the plasmid was designated as PPTC3Z0.1.
  • Plasmid PPTC 3 / 0.1 was digested with EcoRI and a ligation reaction was performed with the 2.0 kb fragment. Similarly, after extracting and purifying plasmid DNA from the transformant, the nucleotide sequence was determined from the BamHI cleavage site by the dideoxy method. ⁇ The 2.O kb fragment was inserted in the forward direction according to the nucleotide sequence. A plasmid was selected and named PPTC 3.2.1.
  • vector DNA and restriction enzymes were purchased from Takara Shuzo. GENECLEAN from BI 0101 was used for recovery of DNA from agarose gel and purification of plasmid from transformant. Also, ligations The ligation reaction was performed using Takara Shuzo's Ligation Kit. The nucleotide sequence was determined using Takara Shuzo M13 Primer RV-N and 7-DEA ZAS equencing Kit.
  • the vector pTVl19N used here has a 1ac promoter, expression of the target protein is induced by adding IPTG to the medium.
  • the protein expressed by PPTC 32.1 has 16 residues at the N-terminal end of Escherichia coli ⁇ -galactosidase at the N-terminal side and about 40 residues in the latter half of the rat C3 ⁇ chain at the C-terminal side. It is a fusion protein with a molecular weight of about 80 kDa.
  • This fusion protein is obtained by transforming the Eschericia coli JM109 strain by the conventional calcium chloride method using the thus obtained PPTC 3 Z2.1. It is produced by culturing BP-3 027) in a nutrient medium.
  • Plasmid PPTC3Z2.1 was cut with EcoRI to obtain a 2.0 kb Ec0RI-Ec0RI fragment. This was further cut with Fokl to obtain a 1.0 kb Ec0RI-F0kI fragment.
  • a DNA was synthesized in which the protein synthesis termination codon TGA was linked to the 14 bp 3 'end of the rat C 3 dg gene, and annealing was performed. It was obtained as a synthetic DNA fragment of F0kI-Ba [eta] ⁇ of the chain.
  • the expression vector pTV111 N (Takara Shuzo) is cut with Ec0RI and Ba ⁇ a, 1.
  • Ligation reaction is performed between three molecules of Okb Eco RI — F okl fragment and F okl — Ban ll synthetic DNA fragment.
  • Escherichia coli JM109 strain ambilicin LB agar is used. The cells were cultured on a medium to obtain a transformant. Plasmid DNA was extracted and purified from Transformant, and the plasmid was named PTRC3Z0.973.
  • Plasmid PTRC3Z0.973 was cut with NcoI and EcoRI. Rat C 3 dg gene Synthesizes DNA with the protein synthesis initiation codon ATG upstream of the 59 'bp at the 5' end of the 5 'end, performs annealing, and performs double-stranded Nc0I1 Obtained as an Ec0RI fragment. A ligation reaction was performed on PTRC 3 Z0.973 cleaved with the synthetic DNA fragment.Escherichia coli JM109 was transformed, transformed, and cultured on LB agar medium containing Ambiciline. I got it.
  • the Brassmid with a 130 bp rat C3 dgc DNA inserted between the protein synthesis start and stop codons was named PTR C3Z 1.035. .
  • JM109 strain was transformed by a conventional calcium chloride method to obtain J9TRC3 / 1.035 (FERMBP-34885).
  • the above transformant was cultured by adding IPTG (isopropylthiogalactoside) to the medium. The expression of the target protein is induced.
  • the protein expressed by PTRC 3 1.035 has a methionine at the N-terminal of the rat C 3 dg 344 residue and a molecular weight of about 39 kDa.
  • E. coli expression vector PTV118N (Takara Shuzo) was digested with EcoRI and KpnI.
  • a 1.2 kb DNA fragment was obtained by agarose gel electrophoresis. The c0RI-KpnI fragment was separated and recovered.
  • a ligation reaction was performed on the cut vector and the 1.2 kb fragment to transform Escherichia coli JM109 strain, which was then cultured on an ambicilin-containing LB agar medium to obtain a transformant. Plasmid DNA was extracted and purified from the transformant, and the plasmid was named PTHC3Z1.240.
  • Plasmid PTHC3Z1.240 was cut with NcoI and EcoRI, and a 4.4 kb NcoI-EcoRI fragment was separated and recovered by agarose gel electrophoresis.
  • a DNA was synthesized in which the human C3 dgc DNA 5'-terminal approximately 70 bp was connected downstream of the protein synthesis initiation codon ATG, and annealing was performed. Was performed to obtain a double-stranded Nc0I-Ec0RI fragment.
  • a ligation reaction was performed on the 4.4 kb PTHC3-1.240-derived fragment and the synthetic DNA fragment, and E. coli JM109 strain was isolated. After the transformation, the cells were cultured on Ambishin Shoyu LB agar medium to obtain a trans-form. The plasmid DNA was extracted and purified from the transformant, and the plasmid was designated as PTHC31.31.37.
  • Plasmid p THC3 / 1.317 was completely cleaved with Kp ⁇ I, then cleaved in a limited (partial) manner with Ec0T14I, and then 4.0 kb of Kpnl-E was determined by agarose gel electrophoresis. Separated from the coT14I fragment and collected.
  • a DNA was synthesized in which the protein synthesis termination codon TAG was linked to about 8 Obp of the human C 3 dgc DNA 3 ′ end, and the protein was synthesized. To obtain two Ec0T1I-KpnI fragments.
  • a ligation reaction was performed on the fragment derived from the PTHC 3 / 1.317 fragment of Okp and the synthetic DNA fragment to transform Escherichia coli strain JM109, and then placed on Ambishin Shayu LB agar medium. The cells were cultured in the same manner to obtain a trans- form. After extracting and purifying plasmid DNA from the transformant, the nucleotide sequence was determined by the dideoxy method from 26 bases upstream of the Nc0I cleavage site and 43 bases downstream of the KpnI cleavage site. It was determined, and a plasmid having a human C3 dgc DNA of 107 bp inserted between the initiation and termination codons was designated as PTHC3 / 1.050.
  • the aforementioned JM109 strain was transformed by a conventional calcium chloride method, and the recombinant microorganism J9THC3Z1.0500 (FERMBP-3 4 8 6) was obtained. Since the vector pTV118N used here has a 1ac promoter, expression of the target protein is induced by culturing the transformant with IPTG added to the medium.
  • the protein expressed by PTHC 3 / 1.050 has a methionine at the N-terminal of human C 3 dg 349 residue and has a molecular weight of about 39 kDa.
  • Example 5 Expression and Expression Protein Purification in E. coli site of inflammation from PLA 2 inhibitory protein
  • PTC 3 no 2.1 (FERMBP-3 0 2 7), J 9 TRC 3 / 1.0 3 5 (FERMBP— 3 4 8 5), or J 9 THC 3 / 1.0 50 (FERMBP-3 4 8 6) ) was cultured overnight in 37 ml of 10 ml of XTY medium containing 50 / ml of ambicilin sodium, then transferred to the same medium of 11 and cultured at 37 • C. After about 5 hours, 100 jt MIPTG was added, and the culture was continued for about 3 hours.
  • each of the anti-sheep magpie IgG antibody-HRP0 complex was diluted 1: 500 in PBS containing 1% BSA. And 4 hours for 2 hours. These were washed three times with PBS, and then stained with a Konica Immunostain HRP kit.
  • the cells were collected by centrifugation at 3,000 G for 5 minutes and suspended in 40 ml of 50 nil TrisHC1 (pH 7.5) buffer. After turbidity, sonication was performed (Kubota INS0NAT0R201M, about 5 minutes at maximum output) to destroy the cells. The sealed body was recovered by centrifugation at 3,00 G for 10 minutes, and 10 ml of 8 M urea- 10 O BIM Tris-chloride buffer (PH 8.0) was added thereto. The plate was kept at room temperature for 30 minutes to solubilize the inclusion body protein.
  • Lyophilized sample Approximately 3 Omg 1% SDS 2 ml 0.2 It was dissolved in M ethyl morpholine acetate (PH 8.0), 2-2-mercaptoethanol (2ME) was added, and the mixture was allowed to stand at room temperature for 30 minutes. After 2 W of 2 ME was newly added and left at room temperature for further 30 minutes, 3 W of 4-bulpyridine was added and reacted at room temperature for 1 hour. Further, 3 / rf of 4-bulbilidine was added thereto, and the mixture was reacted at room temperature for 1 hour. Then, the mixture was immediately subjected to gel filtration HPLC (TSK gel G300SW). Elution was carried out at 50 mM Tris ⁇ HC1-1500 mM NaCl (pH 7.5) at 0.5 rol / min.
  • the amount of protein purified from 1 £ of E. coli culture was about 600 / «.
  • the final sample purified by reversed-phase HPLC was dried using a vacuum centrifugal concentrator, dissolved in 80% acetonitrile-0.1% TFA, and dissolved in a gas phase protein sequencer (Applied Biosystems, Inc., 477). A) was applied and the N-terminal amino acid sequence was determined.
  • N-terminal amino acid sequence of the 8 OkDa fusion protein was found to be the following sequence.
  • Example 7 C 3 or Town fusion protein of Hosuhoribaze eight 2 inhibitory activity
  • Phospholipase A 2 inhibitory activity of gel filtration HPLC fractions # 2 4 were measured.
  • the activity measurement system is 100 ⁇ tris-hydrochloric acid (pH 9.0), 4 mM chloride, 0.1 mM [ 14 C] phosphatidylethanol noramine.
  • Example 8 Rat C 3 or fusion protein against IgE-antigen-lysophosphatidylserine (PS) -dependent activation of rat CTMC
  • the rat C3 or chain fusion protein obtained in Example 5 was added simultaneously with the antigen and lysoPS. Controls were measured for those without added inhibitory protein. As a result, apparent that Hisuta Mi emissions liberated cormorants by shown in FIG. 6 is suppressed, together with results of Example 7, the fusion protein can be inhibited Hisuta Mi emissions released by inhibiting type II PLA 2 It became. In the figure, reference symbols indicate control, and symbol ⁇ indicates this protein.
  • the concentrations required for tranilast, a histamine release inhibitor, to exhibit a significant histamine release inhibitory effect are 10 M each, whereas those of rat C3a ⁇ fusion protein is 2.5 XI 0- 7 M, the protein was found to have a very cooperative anti allergic reaction suppression effect. [Industrial applicability]
  • Plasmid of the present invention a recombinant microbial cell, method of manufacturing the inflammation locally derived phospholipase eight 2 inhibitory protein using it have an allergic reaction suppressive effect
  • mammalian, especially human in ⁇ Rerugi one disease It may be useful in the manufacture of therapeutics.
  • CTGTGTGGGG CTGTCAAATG GCTGATTCTG GAGAAACAGA AGCCAGATGG TGTCTTTCAG 480
  • Fragment type middle part fragment
  • Lys Trp Leu lie Leu Glu Lys Gin Lys Pro Asp Gly Val Phe Gin
  • Sequence type nucleic acid
  • Fragment type middle fragment

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PCT/JP1991/001040 1990-08-03 1991-08-03 Plasmide destine a la production d'une proteine inhibant la phospholipase a2 derivee d'une region enflammee, cellule microbienne, et production et utilisation de ladite proteine WO1992002619A1 (fr)

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KR1019920700763A KR920702418A (ko) 1990-08-03 1991-08-03 염증국소 유래 포스포리파제 a_2저해 단백생산을 위한 플라스미드, 미생물세포 및 그 단백의 제조 및 사용

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JP20516490 1990-08-03
JP2/205164 1990-08-03
JP2/411594 1990-12-19
JP41159490 1990-12-19

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WO1992002619A1 true WO1992002619A1 (fr) 1992-02-20

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EP (1) EP0495995A1 (enrdf_load_stackoverflow)
KR (1) KR920702418A (enrdf_load_stackoverflow)
CA (1) CA2067237A1 (enrdf_load_stackoverflow)
WO (1) WO1992002619A1 (enrdf_load_stackoverflow)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100602403B1 (ko) * 1999-03-03 2006-07-20 닛토덴코 가부시키가이샤 구강 접착 쉬트 및 구강 접착 제제

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7125678B2 (en) 2001-11-23 2006-10-24 Nanogen, Inc. Protein biopolymer markers predictive of type II diabetes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001999A1 (en) * 1989-08-03 1991-02-21 Teijin Limited Phospholipase a2 inhibiting protein originating in inflamed region, production thereof, and gene therefor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Proc. Natl. Acad. Sci. USA, Vol. 82, No. 3, 1985, BRUIJN M. et al. "Human complement component C3:cDNA coding sequence and derived primary structure" P. 708-712. *
Proc. Natl. Acad. Sci. USA, Vol. 87, No. 7, 1990, SUWA Y. et al. "Protein ace ous inhibitors of phospholipase A2 purified from inflammatory sites in rats" P. 2395-2399. *
See also references of EP0495995A4 *
The Journal of Biological Chemistry, Vol. 259, No. 22, 1984, WETSEL et al. "Structure Murine Complement Component C3" P. 13857-13862. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100602403B1 (ko) * 1999-03-03 2006-07-20 닛토덴코 가부시키가이샤 구강 접착 쉬트 및 구강 접착 제제

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EP0495995A1 (en) 1992-07-29
CA2067237A1 (en) 1992-02-04
EP0495995A4 (enrdf_load_stackoverflow) 1994-04-13

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